HRM Methylation Studies Using MeltDoctor ™ HRM Reagents and High Resolution Melt Software v3.0 For detailed instructions and troubleshooting information, refer to the HRM Experiments User Guide (PN 4457847). You can download a PDF version from the Applied Biosystems website at www.appliedbiosystems.com. Note: For safety and biohazard guidelines, refer to the “Safety” section in the HRM Experiments User Guide (PN 4457847). For every chemical, read the SDS and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. 1 Prepare the methylated DNA standards Use the Cells-to-CpG ™ Methylated and Unmethylated gDNA Control Kit, and mix different ratios of 100% methylated and 0% methylated DNA of equal concentration. For example: • To detect high methylation levels, prepare 100%, 75%, 50%, 25%, 10%, and 0% methylation standards. • To detect low methylation levels, prepare 100%, 10%, 5%, 2%, 1%, 0.5%, 0.1%, and 0.0% methylation standards. 2 Treat the samples and methylated DNA standards with bisulfite We recommend that you use a Cells-to-CpG ™ Bisulfite Conversion Kits. For instructions, refer to the Cells-to-CpG ™ Bisulfite Conversion Kit (2x96) Protocol (PN 4449006) or the Cells-to-CpG ™ Bisulfite Conversion Kit (50) Protocol (PN 4448998). 3 Prepare the HRM reactions 4 Amplify and melt the DNA a. Using the real-time PCR instrument software, open and set up the HRM experiment run file: Components Volume for one 20-µL reaction Volume for three 20-µL replicates plus 10% excess MeltDoctor ™ HRM Master Mix 10.0 µL 33.00 µL Primer 1 (5 µM) 1.2 µL 3.96 µL Primer 2 (5 µM) 1.2 µL 3.96 µL Genomic DNA (20 ng/µL) 1.0 µL 3.30 µL Deionized water 6.6 µL 21.78 µL Total reaction volume 20.0 µL 66.00 µL Setup Setting Experiment properties • Experiment type: Quantitation - Standard Curve • Reagents: Other, then select the Include Melt Curve checkbox • Ramp speed: Standard (~ 2 hours to complete a run) Target properties • Reporter: MeltDoctor • Quencher: None Plate layout • Task for negative control wells: • Passive Reference: None Run method • Reaction Volume Per Well: 20 µL • Ramp mode and rate (StepOne ™ and StepOnePlus ™ systems): Select Continuous, then set the ramp rate to 0.3% • Expert Mode (7500 systems): Select the checkbox • (7500 systems) Click Select/View Filters, then select only Filter-1
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HRM Methylation Studies Using MeltDoctor™ HRM Reagents and High Resolution Melt Software v3.0
For detailed instructions and troubleshooting information, refer to the HRM Experiments User Guide (PN 4457847). You can download a PDF version from the Applied Biosystems website at www.appliedbiosystems.com.
Note: For safety and biohazard guidelines, refer to the “Safety” section in the HRM Experiments User Guide (PN 4457847). For every chemical, read the SDS and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.
1 Prepare the methylated DNA standards
Use the Cells-to-CpG™ Methylated and Unmethylated gDNA Control Kit, and mix different ratios of 100% methylated and 0% methylated DNA of equal concentration. For example: • To detect high methylation levels, prepare 100%, 75%, 50%, 25%, 10%, and 0% methylation
standards. • To detect low methylation levels, prepare 100%, 10%, 5%, 2%, 1%, 0.5%, 0.1%, and 0.0%
methylation standards.
2 Treat the samples and methylated DNA standards with bisulfite
We recommend that you use a Cells-to-CpG™ Bisulfite Conversion Kits. For instructions, refer to the Cells-to-CpG™ Bisulfite Conversion Kit (2x96) Protocol (PN 4449006) or the Cells-to-CpG™ Bisulfite Conversion Kit (50) Protocol (PN 4448998).
3 Prepare the HRM reactions
4 Amplify and melt the DNA
a. Using the real-time PCR instrument software, open and set up the HRM experiment run file:
Components Volume for one 20-µL reaction
Volume for three 20-µL replicates plus
10% excess
MeltDoctor™ HRM Master Mix 10.0 µL 33.00 µL
Primer 1 (5 µM) 1.2 µL 3.96 µL
Primer 2 (5 µM) 1.2 µL 3.96 µL
Genomic DNA (20 ng/µL) 1.0 µL 3.30 µL
Deionized water 6.6 µL 21.78 µL
Total reaction volume 20.0 µL 66.00 µL
Setup Setting
Experiment properties
• Experiment type: Quantitation - Standard Curve• Reagents: Other, then select the Include Melt Curve checkbox• Ramp speed: Standard (~ 2 hours to complete a run)
Target properties
• Reporter: MeltDoctor• Quencher: None
Plate layout • Task for negative control wells: • Passive Reference: None
Run method • Reaction Volume Per Well: 20 µL• Ramp mode and rate (StepOne™ and StepOnePlus™ systems): Select
Continuous, then set the ramp rate to 0.3%• Expert Mode (7500 systems): Select the checkbox• (7500 systems) Click Select/View Filters, then select only Filter-1
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HRM Methylation Studies Using MeltDoctor™ HRM Reagents and High Resolution Melt Software v3.0 Quick Reference Card
4 Amplify and melt the DNA (continued)
b. Run the plate:
Note: Adjust the annealing temperature during the amplification to increase or decrease the extent of the PCR bias.
c. Using the instrument system software, analyze the experiment file, verify that the samples amplified, review the peaks in the melt curve, then save the experiment file:
5 Review the high-resolution melt data
a. Using the HRM Software, open the *.eds experiment file from your Real-Time PCR System.
b. Make sure the HRM calibration file that is assigned to the HRM experiment is correct.
c. Add controls to the experiment on the Define screen, then assign the controls to wells on the Assign screen.Note: Set up and define controls in the software for each methylated DNA standard with known percentages of methylated DNA.
Note: For the Control Name, do not use the convention variantN, where N is any number (for example, variant1, variant2, and so on).
Stage Step Temp Time
Holding Enzyme activation 95 °C 10 min
Cycling(40 cycles)
Denature 95 °C 15 sec
Anneal/extend 60 °C 1 min
Melt curve(for StepOne™ and StepOnePlus™ systems only: 0.3% ramp rate)
Denature 95 °C 10 sec
Anneal 60 °C 1 min
High resolution melt 95 °C 15 sec
Anneal 60 °C 15 sec
Plot Example Review the plot
Amplification Plot
Review the Amplification Plot for normal characteristics:• Fluorescence levels that exceed the threshold between cycles 8 and 35• An exponential increase in fluorescence
Note: Note which wells are outliers with CT values that differ from replicates by more than 2.
Melt Curve Verify that the Melt Curve shows no unexpected Tm peaks.
With methylation experiments, you will likely see multiple peaks. The number of peaks in the melt curve is correlated with the number of methylation sites in the amplicon.
Note: Unexpected peaks may indicate contamination, primer dimers, ornon-specific amplification.
Note: The data appear noisy because more data is collected during a high resolution melt curve than during a standard melt curve. The extra data are required for analysis with the HRM Software.
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HRM Methylation Studies Using MeltDoctor™ HRM Reagents and High Resolution Melt Software v3.0 Quick Reference Card
5 Review the high-resolution melt data (continued)
d. For each assay in the plate, select an assay, then specify the analysis settings: • (Optional) Pre- and post-melt regions – Deselect the checkbox and manually enter
temperatures to define the pre- and post-melt regions. • Number of variant groups – Make sure the checkbox is selected so that the software will
automatically determine the number of variant groups.
e. Click Apply Analysis Settings to close the analysis settings and reanalyze the experiment.
f. Save the HRM experiment file.
g. (Optional) View the Derivative Melt Curves, set the pre- and post-melt regions as close as possible to the melting transition region, as in the example below, click Analyze, then savethe changes.
h. Review the plots:
Plot Example Review the plot
Aligned Melt Curves
• Do the melt curves for the methylated DNA standards cluster well? Are there any outliers?
• Which methylated standard melt curves are above and below the melt curves for the unknowns? The estimated percent methylation in the unknowns should lie between the percent methylation in those standards.
Difference Plot
Select a control or any well as the reference, then review: • Variant groups – How many distinct
clusters are displayed? • Outliers – How tight are the curves within
each variant group?
Note: Try selecting different wells as the reference to find the optimal display of the groups.
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HRM Methylation Studies Using MeltDoctor™ HRM Reagents and High Resolution Melt Software v3.0 Quick Reference Card
5 Review the high-resolution melt data (continued)
i. Review the software calls, then omit outliers or change calls as necessary:
Note: For wells with low silhouette scores (below 0.8), review the data.
Note: Remember to click Analyze to reanalyze the data after you omit outliers or change calls.
6 Sequence the variants a. Dilute the PCR products of the selected variants to 0.5–1.5 ng/µL with water. • If you dilute the PCR product >1:20, proceed with the sequencing reactions. • If you dilute the PCR product <1:20, purify the PCR product before you proceed with the
sequencing reactions:
b. Prepare the sequencing reactions:
Sample type Review the software calls
Methylation standard controls
• Variant Call column – Do all of the methylation standard controls have the correct call?
• Silhouette Score column – Are the silhouette scores close to 1.0 (0.8 to 1.0)?
Replicate groups
• Variant Call column – Do all replicates have the same call?• Silhouette Score column – Are the silhouette scores close to 1.0
(0.8 to 1.0)?
All samples The curves for unknown samples should fall within the controls of known %methylation. Estimate the range of %methylation in your unknown samples based on the two flanking control curves.
Component Volume
Diluted PCR product 10 µL
ExoSAP-IT® 2 µL
Total reaction volume 12 µL
Stage Temp Time
1 37 °C 30 min
2 80 °C 15 min
3 4 °C ∞
Component Volume
BigDye® Terminator v1.1 2 µL
Forward primer or reverse primer (3.2 pmol each) 1 µL
HRM Methylation Studies Using MeltDoctor™ HRM Reagents and High Resolution Melt Software v3.0 Quick Reference Card
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